Adaptation to ex vivo culture reduces human hematopoietic stem cell activity independently of the cell cycle

Abstract

Loss of long-term hematopoietic stem cell (LT-HSC) function ex vivo hampers the success of clinical protocols that rely on culture. However, the kinetics and mechanisms through which this occurs remain incompletely characterized. In this study, through time-resolved single-cell RNA sequencing, matched in vivo functional analysis, and the use of a reversible in vitro system of early G1 arrest, we defined the sequence of transcriptional and functional events that occur during the first ex vivo division of human LT-HSCs. We demonstrated that the sharpest loss in LT-HSC repopulation capacity happens early on, between 6 and 24 hours of culture, before LT-HSCs commit to cell cycle progression. During this time window, LT-HSCs adapt to the culture environment, limit the global variability in gene expression, and transiently upregulate gene networks involved in signaling and stress responses. From 24 hours, LT-HSC progression past early G1 contributes to the establishment of differentiation programs in culture. However, contrary to the current assumptions, we demonstrated that the loss of HSC function ex vivo is independent of cell cycle progression. Finally, we showed that targeting LT-HSC adaptation to culture by inhibiting the early activation of JAK/STAT signaling improves HSC long-term repopulating function ex vivo. Collectively, our study demonstrated that controlling early LT-HSC adaptation to ex vivo culture, for example, via JAK inhibition, is critically important to improve HSC gene therapy and expansion protocols.

Graphical abstract

Figure 1.

Kinetics of cell cycle progression, survival, and loss of long-term repopulation capacity of LT-HSCs during ex vivo culture. (A) The cumulative quiescence exit kinetics of GT_mPB LT-HSCs are shown, determined by phosphorylation of Rb (pRb; at Ser 807-811) using flow cytometry analysis. The curve is a least-squares sigmoidal fit with half maximal effective concentration (EC₅₀) = 24.7 hours; there are n = 3 biological replicates for 0 hours, 24 hours, 62 hours, and 72 hours and n = 4 biological replicates for 6 hours. Standard error ± 5.186; R² = 0.9797. (B) The cumulative quiescence exit kinetics of EXPER_CB LT-HSCs are shown, determined by pRb (at Ser 807-811) using flow cytometry analysis. The curve is a least-squares sigmoidal fit with an EC₅₀ = 24.72 hours; there are n = 3 biological replicates for 0 hours, 6 hours, 24 hours, and 48 hours and n = 4 biological replicates for 72 hours. Standard error ± 3.944; R² = 0.9844. (A-B) The dashed line indicates the EC₅₀ at the time of quiescence exit. (C) Cell cycle phase assignment of GT_mPB LT-HSCs determined by pRb/DAPI flow cytometry analysis. Equivalent repeats as in panel A. (D) The cell cycle phase assignment of EXPER_CB LT-HSCs determined by pRb/DAPI flow cytometry analysis. Equivalent repeats as in panel B. (E) The cumulative first-division kinetics (excluding dead cells) of GT_mPB LT-HSCs are shown. The curve is a least-squares sigmoidal fit. Representative examples are shown (n = 3 biological replicates). The dashed line indicates the EC₅₀. The EC₅₀ = 64.83 hours; 95% confidence interval (CI) = 62.83-66.87; R² = 0.9976. (F) The cumulative first-division kinetics (excluding dead cells) of EXPER_CB LT-HSCs are shown. The curve is a least-squares sigmoidal fit. Representative examples shown (n = 4 biological replicates). EC₅₀ = 55.44 hours; 95% CI = 54.28-56.70; R² = 0.9995. (G) The time to first-division kinetics summary of LT-HSCs cultured in (E) GT_mPB (F) and EXPER_CB systems (n = 3 biological replicates for GT_mPB; n = 4 biological replicates for EXPER_CB). Unpaired t tests are shown. (H) The percentage of LTRC in GT_mPB CD34⁺CD38⁻ cells at each time point, as determined by LDA analysis in the transplanted population ± 95% CIs is shown. The LTRC frequency estimates for GT_mPB are as follows: 0 hours, 1 in 939 (29 mice); 6 hours, 1 in 1211 (21 mice); 24 hours, 1 in 3371 (23 mice); 62 hours, 1 in 2510 (23 mice). Extreme Limiting Dilution Analysis (ELDA) statistical tests are shown. The data are shown in supplemental Table 1. (I) The percentage of LTRC in LT-HSCs cultured in EXPER_CB systems at each time point, based on LDA analysis in the transplanted population ± 95% CIs is shown. The LTRC frequency estimates for EXPER_CB are as follows: 0 hours, 1 in 14.8 (31 mice); 6 hours, 1 in 15.2 (19 mice); 24 hours, 1 in 80.6 (31 mice); and 72 hours, 1 in 293.7 (40 mice). ELDA statistical tests are shown. The data are shown in supplemental Table 1. (J) The survival of LT-HSCs cultured in GT_mPB systems, as determined by Annexin-V/7-AAD flow cytometry, is shown for n = 3 biological replicates at each time point. The mean ± SD is shown. (K) Survival of LT-HSCs cultured in EXPER_CB systems, as determined by Annexin-V/7-AAD flow cytometry, is shown for n = 3 biological replicates at each time point. Paired t tests are shown for the 6 hours and 24 hours comparison. The mean ± SD is shown. SD, standard deviation.

Figure 2.

The dynamics of gene expression over the first division of LT-HSC ex vivo at single-cell resolution. (A) Uniform manifold approximation and projection (UMAP) of 429 single EXPER_CB LT-HSCs over a time course of 0, 6, 24, and 72 hours (n = 2 independent experiments). The UMAP was generated using the Seurat 4 pipeline after cell cycle regression. (B) A 2-dimensional pseudotime rank plot of EXPER_CB LT-HSCs over the time course generated after cell cycle regression. (C) Transcriptional allocation of cell cycle status at different time points during EXPER_CB LT-HSC culture; n = 429 single cells. (D) The number of differentially expressed genes (false discovery rate < 0.05) at each time point in comparison with the 0 hours time point. Upregulated genes are shown in blue, and downregulated genes are shown in brown. A full list of genes is available in supplemental Table 4. (E) The broad patterns of gene expression that were identified over the time course (8966 genes classified after filtering by the DEG report algorithm). Numbers indicate the percentage of genes that showed the specific patterns of gene expression displayed to the right of the bar. (F-J) GSVA score of c2 curated pathways showing the following specific expression patterns: (F) continuous up; (G) continuous down; (H) transient up; (I) transient down; and (J) up later than 6 hours. The GSVA score that was calculated per single cell with a line at the median and upper and lower whiskers indicating the 25th and 75th percentile of expression. (K) The GSVA scores of indicated published gene signatures, representative of specific HSCs and progenitor cell (HSPC) subsets. The median and interquartile range are shown. ∗P < .001. (L) The scEntropy value at each time point is shown (calculated for both batches combined; Wilcoxon rank sum test shown; 0 vs 6 hours; P = .835). The median and interquartile range are shown. ∗P < .001. (M) Shows the number of maximally variable genes (MVGs) at each time point (supplemental Methods; 2792 genes total). (N) Shows selected biological pathways that are significantly enriched from MVGs (–log₁₀[adjusted P value] < .05). A full list of the pathways available in supplemental Table 7. CMP, common myeloid progenitor; GMP, granulocyte monocyte progenitor; MEP, myeloid erythroid progenitor.

Figure 3.

Transcriptional effects of preventing progression past early G₁ during ex vivo culture of LT-HSCs. (A) The cumulative first-division kinetics (excluding dead cells) of UNTR/PD-treated LT-HSCs cultured in a GT_mPB (dark red and light red) or EXPER_CB (dark blue and light blue) system. The curve is a least-squares sigmoidal fit. A representative example is shown (n = 3 UNTR or PD–matched biological replicates: n = 2 UNTR or PD–treated matched biologic replicates). The dashed line indicates the EC₅₀ time to first division. UNTR and PD-treated (200 nM) cells are shown. (B) The divided single cells as a proportion of the total alive cells at 96 hours are shown (EXPER_CB: n = 5 biological replicates, n = 2 UNTR or PD–treated matched biological repeats; GT_mPB: n = 3 matched biological replicates). Paired t tests are shown. (C) Representative example of the flow cytometry plot for pRb (at Ser 807-811) and DAPI staining on 72 hour EXPER_CB cultured UNTR (left) or PD-treated (right) LT-HSCs. (D) Quantification of pRb⁺ (as a percentage of viable cells) in 62 hour GT_mPB cultured UNTR or PD-treated LT-HSCs (n = 3 UNTR or PD-treated matched biological replicates; no lentiviral vector [LV] transduction) or 72 hour EXPER_CB cultured LT-HSCs (n = 3 UNTR or PD-treated matched biological replicates). Paired t tests are shown. (E) UMAP visualization of scRNA-seq of 954 LT-HSCs from the indicated culture conditions (EXPER_CB, 536 single cells; GT_mPB, 418 single cells). Cell cycle regression was applied. (F) The 2-dimensional pseudotime density rank plot of single cells as shown in (E). Cell cycle regression was applied. (G) Pearson correlation coefficient estimates for the comparison of the median expression value of 10903 genes at different time points or conditions for the EXPER_CB data set (union of all differentially expressed genes between any 2 UNTR time points and between PD-treated and UNTR conditions) (available in supplemental Table 5). (H) Pearson correlation coefficient estimates for the comparison of the median expression value of 5469 genes at different time points or conditions for the GT_mPB data set (union of all differentially expressed genes between any 2 UNTR time points and between PD treated and UNTR conditions) (available in supplemental Table 5). (I) Selected Reactome pathways (FDR < 0.05) enriched between PD-treated and UNTR LT-HSCs cultured for matched durations of 24 hours in EXPER_CB (purple), 72 hours in EXPER_CB (pink), and 62 hours in GT_mPB (brown) systems. The full DeSeq2 results and Reactome pathway enrichment are available in supplemental Table 8.

Figure 4.

Preventing progression past early G₁ during ex vivo culture of LT-HSCs dampens the establishment of differentiation programs but does not affect the loss of long-term repopulation capacity. (A) GSVA scores for the indicated lineage gene expression signatures from Laurenti et al at the indicated time points for the LT-HSC culture. The GSVA score is generated per cell, and the line indicates the median. EXPER_CB, n = 536 single cells; GT_mPB, n = 418 single cells. (B) The cell diameters of single EXPER_CB cultured LT-HSCs (n = 2 biological replicates representing n = 469 total single cells). Unpaired t tests are shown. ∗P < .001. (C) Tetramethylrhodamine (TMRM) staining of bulk EXPER_CB cultured LT-HSCs (n = 5 biological replicates for 0 hours; n = 4 matched biological replicates for UNTR or PD-treated cells for 24 hours; n = 3 matched biological replicates for UNTR or PD-treated cells for 48 hours). Unpaired t tests are shown. (D) Workflow of in vivo transplantation of EXPER_CB cultured LT-HSCs (24 hours and 72 hours) and CD34⁺/CD38⁻ cells cultured in the GT_mPB system (62 hours). UNTR or PD-treated cells were transplanted in matched cell dose experiments. (E) Graft size (percentage of human CD45⁺⁺ and GlyA⁺) at 18 weeks after transplantation of UNTR or PD-treated EXPER_CB LT-HSCs cultured for 72 hours (n = 5 biological experiments; the graph is representative of engrafted mice only; n = 42 PD mice; n = 38 UNTR mice). Two-way analysis of variance (ANOVA) with Sidak multiple comparisons performed (50 cells UNTR vs 50 cells PD; P = .9552; 300 cells UNTR vs 300 cells PD; P = .4084; 700 cells UNTR vs 700 cells PD; P = .971). (F) Graft size (percentage of human CD45⁺⁺ and GlyA⁺) at 18 weeks post transplantation of mPB CD34⁺CD38⁻ cells after GT protocol culture for 62 hours including LV transduction (n = 3 biological replicates; the graph is representative of engrafted mice only; n = 25 mice UNTR; n = 26 mice PD-treated). Two-way ANOVA with Sidak multiple comparisons performed (all cell doses UNTR vs PD; P > .9). (G) The percentage of LTRC in EXPER_CB cultured LT-HSCs UNTR or PD-treated, determined at 24 hours (n = 31 mice UNTR; n = 30 mice PD) and 72 hours (n = 42 mice PD; n = 40 mice UNTR). Numerical estimates for LTRC frequency available in supplemental Table 2. ELDA statistics (24 hours UNTR vs 24 hours PD; P = .405; 72 hours UNTR vs PD; P = .426). (H) LDA of secondary transplantation experiment from EXPER_CB cultured LT-HSCs UNTR or PD-treated from the 72 hour primary mice cohort. Secondary animals were transplanted with sorted CB CD45⁺⁺ from primary recipients (n = 20 mice total; 10 UNTR, 10 PD; n = 1 experiment) (supplemental Table 10). An ELDA statistical test was performed (P = .190). (I) LDA of secondary transplantation experiment from GT_mPB UNTR or PD-treated from the 62 hour primary mice cohort. Secondary animals were transplanted with whole mouse bone marrow (BM) isolated from primary recipients (n = 21 mice total; UNTR = 11 mice; PD = 10 mice; n = 1 experiment) (supplemental Table 10). An ELDA statistical test was performed (P = .860). (D) was created with BioRender.com (license agreement, KE26QKHW50).

Figure 5.

RUX treatment improves serial replating ability and self-renewal capacity of cultured HSCs. (A) GSVA score of the Kyoto Encyclopedia of Genes and Genomes (KEGG) JAK/STAT signaling pathway gene-set (same as Figure 2H). The GSVA score was calculated per single cell with the line indicating the median and the upper and lower whiskers indicating the 25th and 75th percentile of expression. (B-C) Serial replating of human mPB HSC/MPPs (CD34⁺CD38⁻CD45RA⁻) cultured for 72 hours in EXPER conditions (B) or for 62 hours in GT conditions (C). Graphs (upper panels) show the fold change in the colony number in comparison with DMSO from primary (2 week), secondary (4 week), and tertiary (6 week) plating. There are n = 3 mPB biological replicates in (B) and n = 4 mPB biological replicates in (C); the individual donors are indicated by shapes. The mean ± SD are shown. Tables (lower panels) report the statistics from a generalized linear mixed-effects model (glmer) analysis performed with raw colony counts. Tukey corrected P values for pairwise comparisons with DMSO with P < .05 are shown (supplemental Table 12 contains all comparisons and raw data). (D) Representative image of wells from tertiary replating experiment of mPB HSC/MPPs cultured for 72 hours in EXPER conditions (upper panel) or for 62 hours in GT conditions (lower panel) treated with either DMSO (left) or 10 nM RUX (right). Circles indicate manually scored colonies. Images brightened by 17%. (E-G) Serial replating of human mPB HSC/MPPs cultured for 62 hours in GT conditions. Graphs (upper panels) show fold change in colony number in comparison with DMSO from tertiary (6 week) plating in the conditions indicated as follows: (E-G) RUX (10 nM); (E) 0 hours fresh HSC/MPPs; (F) UM171 (35 nM), low TPO (20 ng/mL); (G) the pan-caspase inhibitor (CASi) Z-VAD(OH)-FMK (100 nM); (E,G) n = 3 mPB biological replicates; (F) n = 5 mPB biological replicates. Individual donors indicated by shapes and matching across (E-G). The mean ± SD are shown. Tables (lower panels) report the statistics from the glmer analysis with fitting of raw colony counts. Tukey corrected P values for pairwise comparisons of EM means with P < .05 are shown (supplemental Table 12 contains all comparisons and raw data). (H) Workflow of in vivo transplantation of mPB CD34⁺CD38⁻ cells cultured in GT conditions for 62 hours with LV transduction and RUX (10 nM) or DMSO. Secondary transplantations were performed from whole BM of engrafted mice. (I) Graft size (percentage of human CD45⁺⁺ and GlyA⁺) in the BM at 18 weeks post transplantation of mPB CD34⁺CD38⁻ cells cultured for 62 hours in GT conditions with RUX (10 nM) or DMSO. n = 6 biological replicates; the graph shows representative of n = 68 engrafted mice (n = 34 DMSO; n = 34 RUX). Two-way ANOVA with Sidak multiple comparisons was performed (30 000 cells DMSO vs 30 000 cells RUX; P = .005; all other doses DMSO vs RUX; P > .9). (J) The log-fraction plot of the limiting dilution model fitted to data in supplemental Table 14. Indicates the percentage of LTRC estimates from secondary transplantation experiments. Whole BM of engrafted mice from primary transplants was transplanted in NSG-SGM3 mice and analyzed 8 weeks posttransplantation (n = 1 experiment; n = 35 mice). Slope indicates the log-active fraction. The dotted line shows the 95% CI. Zero negative response indicated by triangle. Panel H was created with BioRender.com (license agreement UR26QKHL5H).